Medical use of tectorigenin in treatment of chicken necrotic enteritis

ABSTRACT

Provided is use of tectorigenin in the preparation of a medicament for treatment of chicken necrotic enteritis. Using tectorigenin for treating chicken necrotic enteritis can significantly reduce the degree of pathological changes in the intestinal tract of chickens with necrotic enteritis, and has a good therapeutic effect on chicken necrotic enteritis caused by Clostridium perfringens.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority of Chinese Patent Application No.201810470739.7, filed with the China National Intellectual PropertyAdministration on May 17, 2018, and entitled with “MEDICAL USE OFTECTORIGENIN IN THE TREATMENT OF CHICKEN NECROTIC ENTERITIS”, and thecontent of which is incorporated herein by reference in its entirety.

FIELD

The present disclosure discloses the medical use of tectorigenin in thetreatment of chicken necrotic enteritis, and relates to the applicationof tectorigenin in the preparation of a medicament for the treatment ofchicken necrotic enteritis, which belongs to the field of medicamenttechnology.

BACKGROUND

Clostridium perfringens is an anaerobic Gram-positive bacillus andwidely present in soil, water, food, human and animal feces, livestockfeed and intestinal tract of animals in nature. Clostridium perfringensis a normal intestinal flora of human and animals and belongs toconditional pathogen. Currently, Clostridium perfringens are dividedinto five serotypes A-E according to different exotoxins they secreted.Poultry are mainly infected with Clostridium perfringens type A and C,especially type A. After infecting poultry, Clostridium perfringensmainly cause necrotic enteritis, the symptoms of which are depression,fluffy and untidy plume, reduced or no appetite, bloody stool andseverely reduced production performance for chickens. Lesions are mainlyin small intestine, which is characterized by thinning of intestinalwall, and focal or piece of necrosis and shedding of intestinal mucosa.According to relevant research statistics, the economic loss caused bynecrotic enteritis in global poultry industry has reached billions ofdollars, which has seriously endangered the development of poultryindustry.

The adhesion colonization of Clostridium perfringens in intestinal tractis mainly mediated by its Type IV pili (TFP). TFP is a kind of movementorgan that exists on cell membrane of bacteria. TFP endows Clostridiumperfringens an ability of moving in its natural habitat and on thesurface of semi-solid media such as soft and hard agar and the like.This special movement is referred as Gliding motility. According toresearch, bacterial functions, including adhesion colonization, glidingmotility and biofilm formation and the like of Clostridium perfringensin intestinal tract, are all somewhat related to TFP. By inhibitingfunctions of TFP of Clostridium perfringens, it's possible to interferewith adhesion colonization of bacteria in intestinal tract, so as toachieve the purpose of resisting Clostridium perfringens infection.

Adding antibiotics to feed is a main approach for the prevention andtreatment of poultry infected with necrotic enteritis. However, problemsof bacterial resistance and antibiotic residue caused by the large-scaleuse of antibiotics poses a serious threat to public health. Therefore,development of safe and effective antibiotic alternatives isincreasingly becoming a hotspot in research. Natural compounds haveadvantages such as low toxicity, abundant sources, and wide range ofsafety and the like, and are important resources for screeningantibiotic alternatives.

Tectorigenin is a naturally-occurring isoflavonoid compound and mainlypresent in roots and stems of Iridaceae iris L. and Belamcanda adansplants. Current researches have showed that tectorigenin has biologicalactivities such as anti-inflammatory, anti-cancer, hepatoprotective,antioxidant and estrogen-like effects, and the like. In recent years, alarge amount of literature has reported pharmacological activities oftectorigenin. However, up to now, there is no relevant research on usingtectorigenin for the treatment of chicken necrotic enteritis caused byClostridium perfringens around the world.

SUMMARY

The present disclosure provides medical use of tectorigenin in thetreatment of chicken necrotic enteritis, and tectorigenin has a goodtherapeutic effect on chicken necrotic enteritis caused by Clostridiumperfringens.

The molecular structure of the tectorigenin in the present disclosure isas follows:

The present disclosure verifies that tectorigenin has a protectiveeffect on chicken necrotic enteritis caused by Clostridium perfringensinfection through inhibition test of gliding motility of Clostridiumperfringens, inhibition test of biofilm formation, and chicken necroticenteritis model test.

I. Inhibition Test of Gliding Motility

The standard strain of Clostridium perfringens ATCC13124 was culturedanaerobically in liquid BHI medium at 37° C. for 12 h, and then 300 μlof the culture was taken and continued to be cultured anaerobically inliquid BHI at 37° C. for 5 h. 1 ml of the culture was taken andcentrifuged at 10,000 g to discard supernatant. The bacterium obtainedwere resuspended in 500 μl of BHI liquid medium to double theconcentration. Subsequently, 5 μl of the concentrated bacterial solutionwas taken and added dropwise respectively to 0.7% BHIA plates containing8 μg/ml and 32 μg/ml tectorigenin, which were prepared previously anddried in an oven at 37° C. for 1 h. The plates were culturedanaerobically at 37° C. for 48 h, and images were collected (see FIG.1). The result showed that 8 μg/ml and 32 μg/ml tectorigenin can bothsignificantly inhibit the gliding motility of Clostridium perfringens onBHIA, and the effect of 32 μg/ml tectorigenin was more remarkable.

II. Inhibition Test of Biofilm Formation

Clostridium perfringens were cultured anaerobically in liquid BHI at 37°C. for 12 h, and then centrifugation was conducted to discardsupernatant. The bacterium obtained were washed once with sterile PBS,and then resuspended in TSB liquid medium and adjusted toOD_(600 nm)=0.1. 400 μl of adjusted bacteria culture was taken and addedto 24-well plate. Subsequently, different concentrations of tectorigeninwere respectively added to the bacterial culture, such that the finalconcentration of tectorigenin in 24-well plate was 4 μg/ml, 8 μg/ml, 16μg/ml and 32 μg/ml respectively. Additionally, a positive control groupwith addition of 0.4 μl filter-sterilized DMSO (without addition oftectorigenin) and a negative control group of 400 μl liquid TSB mediumwere prepared, and added respectively to 24-well plate to anaerobicallyculture at 30° C. for 5 days. The supernatant was then discarded byaspiration. The plate was washed twice with sterilized PBS, and dried atroom temperature for 30 min. The plate was added with 400 μl offilter-sterilized 1% crystal violet, and incubated at room temperaturefor 30 min. Crystal violet was then discarded. The plate was washedtwice with sterilized PBS again. 33% acetic acid was added in each wellto dissolve the crystal violet. After pipetting and mixinghomogeneously, 300 μl of the solution was taken and added to 3 separatewells with 100 μl per well of 96-well plate respectively. The absorbancewas measured at 570 nm using a microplate reader, and the biofilmformation rate was calculated based on the absorbance of crystal violet.The result showed that tectorigenin can effectively inhibit the biofilmformation of Clostridium perfringens (see FIG. 2).

III. Therapeutic Study of Chicken Necrotic Enteritis Model Test

1. Chicken Necrotic Enteritis Model

17-day-old male Arbor Acre broiler chickens were orally administered 0.5ml (1×10⁹ CFUs) of Clostridium perfringens ATCC13124 culture resuspendedin sterile PBS. On the next day (18-day-old), the bacteria solutionhaving the same bacteria amount was administered again to challengebacteria, so as to establish a model of chicken necrotic enteritis. Thechickens in the healthy group were orally administered 0.5 ml of sterilePBS as a control.

2. Intestinal Lesion Score Test

Chickens were orally administered with 25 mg/kg of tectorigenin(dissolved in sterilized PBS) once every 12 h according to body weightof chickens 4 h before oral bacteria challenge infection (17-day-old).In addition, a positive control group without medicament treatment and ahealthy control group without bacteria challenge infection wereprepared. The chickens in the positive control group and healthy controlgroup were weighed in the same way and were orally administeredcorresponding volume of sterile PBS according to body weight. Each ofthe above three groups has 10 chickens, and the treatment group has atotal of 5 days of treatment with medicament administration (treatmentto 21-day-old). All chickens were sacrificed at 21-day-old by cervicaldislocation. Autopsy was performed and the duodenum to the jejunum wastaken out. The pathological changes in the intestinal tract wereobserved visually, and intestinal injury scoring was performed accordingto the degree of intestinal injury (see FIG. 3). The intestinal injuryscoring was based on a 0-6 scores system: 0 score (with no obviousinjury), 1 score (the bowel wall becoming thin and brittle), 2 scores(with 1-5 necrotic lesions), 3 scores (with 6-15 necrotic lesions), 4scores (with 16 or more necrotic lesions), 5 scores (with a 2-3 cm piecein length of necrosis), 6 scores (with a large area of diffusenecrosis). In addition, diseased materials was collected in theabove-mentioned small intestine, and was subjected to HE staining tomake pathological sections. The histopathological changes were observedunder a microscope (see FIG. 4).

During the bacteria challenge, all broiler chickens were weighed daily,and the average body weight and daily weight gain were calculated toinvestigate the effect of tectorigenin on the daily weight gain ofbroiler chickens with necrotic enteritis after treating withtectorigenin (see FIG. 5).

The result of intestinal lesions scoring showed that after the treatmentwith tectorigenin, the degree of pathological changes of the necroticenteritis in the intestinal tract of chickens was significantly reduced.Histopathological observations showed that the intestinal villus ofsmall intestine for broiler chickens in the healthy group were intact instructure, without bleeding and inflammatory exudation; while theintestinal villus of small intestine for broiler chickens in the controlgroup were necrotic and dissolved, incomplete in structure, andaccompanied by massive bleeding. After the treatment with tectorigenin,the histopathological injury described above was significantlyalleviated. The result of daily weight gain showed that the daily weightgain of broiler chickens in the treatment group was similar to that ofbroiler chickens in the healthy group, both of which were significantlyhigher than that of the infected group.

The above results indicated that tectorigenin has a good protectiveeffect on chicken necrotic enteritis caused by Clostridium perfringens.

The positive effect of the present disclosure is to provide a newmedical use of tectorigenin for the treatment of chicken necroticenteritis, which can significantly reduce the degree of pathologicalchanges in the intestinal tract of chickens with necrotic enteritis, andhas a good therapeutic effect on chicken necrotic enteritis caused byClostridium perfringens.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an image showing that tectorigenin can significantly inhibitthe gliding motility of Clostridium perfringens on BHIA;

FIG. 2 is an image showing that tectorigenin can effectively inhibit thebiofilm formation of Clostridium perfringens;

FIG. 3 is an image showing the scoring of intestinal injury performedaccording to the degree of intestinal injury;

FIG. 4 is a picture showing the histopathological changes observed undera microscope;

FIG. 5 is an image showing the effect of tectorigenin treatment on thedaily weight gain of broiler chickens with necrotic enteritis.

DETAILED DESCRIPTION Example 1

Provided is use of tectorigenin in the preparation of a medicament forthe treatment of chicken necrotic enteritis, and any pharmaceuticallydosage form prepared from tectorigenin as an active ingredient.

Example 2

The chicken necrotic enteritis treated with tectorigenin is necroticenteritis caused by bacteria.

Example 3

The chicken necrotic enteritis treated with tectorigenin is necroticenteritis caused by Clostridium perfringens.

1. A method for treating chicken necrotic enteritis, comprisingadministering to chicken a therapeutically effective amount oftectorigenin.
 2. The method according to claim 1, wherein the chickennecrotic enteritis is necrotic enteritis caused by bacteria.
 3. Themethod according to claim 1, wherein the chicken necrotic enteritis isnecrotic enteritis caused by Clostridium perfringens.
 4. Anypharmaceutically acceptable dosage form prepared from tectorigenin as anactive ingredient.